Diversity, Leishmania detection, and blood meal sources of sand flies from Iguatama, Minas Gerais, Brazil

This study investigated the sand fly fauna of the municipality Iguatama, in the Midwest Region of Minas Gerais state, Brazil, including Leishmania infection rates and blood meal sources. Sand flies were collected during four periods over the course of a single year, encompassing both dry and rainy seasons, using CDC light traps placed in peridomiciles where dogs were seropositive for visceral leishmaniasis (VL). A total of 762 sand fly specimens, representing 12 species across seven genera, were collected. Lutzomyia longipalpis was the most abundant species, comprising 57.6% of the collected specimens, followed by Nyssomyia neivai (19.6%) and Nyssomyia whitmani (10.5%). Species richness and diversity varied among collection periods, with the highest diversity observed in January 2019. Molecular analysis detected Leishmania DNA in 12.5% of the sand fly specimens, with Le. infantum being the predominant species. Blood meal analysis revealed feeding on multiple vertebrate species, including humans, rats, dogs, and chickens. The presence of Leishmania DNA in sand flies, and the identification of human blood meals, highlight the potential role of these species in VL transmission. These findings underscore the importance of continued surveillance and control measures to prevent the spread of VL and reduce transmission risk in the region.


Introduction
Sand flies (Diptera: Psychodidae), are of considerable ecological importance due to their crucial role in the transmission of various pathogens, including protistan parasites of the genus Leishmania Ross, 1903, the causative agent of leishmaniasis [1].This disease, with both cutaneous (CL) and visceral (VL) forms, is highly endemic in Brazil and geographically widespread [2].Leishmaniasis incidence rates in Brazil have had a significant impact on public health, with numerous reported cases and the involvement of multiple Leishmania species [3].

Study area, collection, and identification of sand flies
The municipality of Iguatama (20˚10'26" S, 45˚42'39" W), located in the Midwest Region of Minas Gerais State (MG), Brazil (Fig 1), is characterized by a tropical savannah climate, as classified by the Ko ¨ppen-Geiger system [11].The natural vegetation of the region is predominantly Cerrado, and the rainy season typically extends from October to March, while the dry season lasts from April to September.
Sand flies were collected during four periods over the course of August 2018 and September 2019, with two collection campaigns in the dry season (May 2019 and September 2019) and two others in the rainy season (August 2018 and January 2019).Two CDC light traps were placed in 29 peridomiciles that housed dogs seropositive for VL, treated with Miltefosine [12,13], for two consecutive nights.All peridomiciles exhibited similar characteristics, including fruit trees and domestic animals (dogs, cats, and chickens).Collecting was conducted in accordance with the Sistema de Autorizac ¸ão e Informac ¸ão em Biodiversidade-SISBIO (License N˚15237-2), the Brazilian environmental agency.
The collected sand flies were stored at -20˚C in 1.5mL tubes containing 70% ethanol solution until dissection.Female specimens were dissected on glass slides containing phosphatebuffered saline, with the head and last three abdominal segments being removed for morphological identification of species.The remaining thorax and abdomen were individually stored dry at -20˚C until DNA extraction.Male specimens were mounted on glass slides using Berlese liquid.
All sand flies were identified following the classification proposed by Galati [14].Females of Evandromyia cortelezzii (Brethes, 1923) were identified to the species level since no other species of the Cortelezzii complex [15] were collected.The abbreviations for sand fly genera followed Marcondes [16].

Ecological assessments of the sand fly fauna
Ecological assessments of the sand fly fauna were conducted to analyze population patterns and diversity indices using Microsoft Excel (Office 2016).The cumulative number of species was calculated to estimate species richness over the study period and assess the adequacy of sampling efforts with CDC light traps.Non-linear logarithmic regression analysis was performed to examine the potential influence of extrapolated hypothetical collections on species richness.Species richness was also estimated using the classical formula of the Chao1 Index.Ecological analyses of the collection sites utilized Shannon (H) and Simpson (D) diversity indices, as well as the Pielou Evenness Index (J) [17,18].Constancy Index was used to evaluate the frequency of each species throughout the study period [19].All ecological indices were calculated using EstimateS software version 9.1.0.

Molecular detection of Leishmania
For molecular detection of Leishmania DNA, whole DNA of non-engorged individual female sand flies was extracted using the Gentra Puregene Cell and Tissue Kit (Qiagen, Valencia, CA), following the manufacturer's protocol.The extracted DNA was then stored at -20˚C until further molecular assays.Negative control groups consisting of Lutzomyia longipalpis males were included during DNA extraction to avoid potential cross-contamination.Instruments and working areas were decontaminated using DNAZap (Ambion Life Technologies, Inc.) to ensure accuracy of results.
A PCR assay was conducted to detect the presence of Leishmania DNA, using primers 150 (5' GGGKAGGGGCGTTCTSCGAA 3') and 152 (5' SSSWCTATWTTACACCAACCCC3'), targeting a conserved region of the kinetoplast DNA minicircle, following conditions previously described [20,21].A positive control was included in all PCR assays, consisting of the reference strain of Leishmania infantum (MHOM/BR/1974/PP75), along with a negative control of nontemplate samples.PCR-positive products were purified using the ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher, California, USA), and subsequently subjected to Sanger sequencing [22].All obtained sequences were analyzed using Finch TV software (Geospiza, Inc., Seattle, USA), and amplicons were compared with sequences from the GenBank database using the BLAST tool.

Blood meal analysis
Blood meal analysis was conducted to identify the source of engorged female sand flies' blood meals.Engorged female sand flies were dissected as previously described for non-engorged females.The remaining parts were stored dry at -20˚C until DNA extraction.Whole DNA was extracted using the QIAamp1 Blood Kit (Qiagen, USA), following the manufacturer's protocol.Precautions were taken to prevent cross-contamination during DNA extraction [23].A PCR assay was conducted using the set of primers cytb1 (5'-CCATCCAACATCTCAGCATGAT GAAA-3') and cytb2 (5'-GCCCCTCAGAATGATATTTGTCCTCA-3') targeting a 359bp fragment of the Cytochrome B gene (cytb) to identify the source of blood meals, following conditions previously described [24].Positive controls consisted of DNA extracted from a blood sample of Gallus gallus, while non-template samples were included as negative controls.PCR-positive products were purified using the ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher, California, USA) and subsequently subjected to Sanger sequencing.The obtained sequences were analyzed and compared to those deposited in the GenBank database.

Results
A total of 762 sand fly specimens were collected, comprising 12 species from seven genera.The predominant species was Lu. longipalpis, which accounted for 57.6% of all collected specimens, followed by Ny. neivai and Ny.whitmani, which represented 19.6% and 10.5%, respectively.These three species together represented 88% of all captured sand flies (Table 1).The highest species diversity was observed in January 2019, with 10 species (H = 1.35;D = 0.35), followed by May 2019 with eight species (H = 1.31;D = 0.31), and August 2018 with six species (H = 0.92; D = 0.53).The species accumulation curves indicated that richness reached its maximum after the third collection, with stabilization of the logarithmic curve.However, additional species were expected in the study area, suggesting a need for increased sampling effort (R 2 = 0.9492) (Fig 2).The estimated real richness value, as indicated by the Chao1 Index, further supported the presence of additional species beyond those observed (Observed Richness = 12 species; Chao1 value = 13.5;SE ± 2.6) (Table 1).The Pielou Index indicated that the high abundance of Lu. longipalpis, Ny. neivai, and Ny.whitmani had a significant impact on the homogeneity of the fauna throughout all months of collection, ranging from 0.51 (August 2018) to 0.63 (May 2019) (Table 1).
Of the total of 12 species, only five (41.6%) were collected in all months and were classified as highly abundant.These included the main vector species Lu. longipalpis, Ny. neivai, and Ny.whitmani.Two species, Pi. christenseni and Sc.sordellii, were considered frequent, while the remaining five species were categorized as occasional (Table 1).
A total of 61 engorged females were tested for blood source identification.Among them, 24 samples (39.3%), representing six species, tested positive by cytb-PCR for blood meal identification.Sanger sequencing revealed that the females had fed on four species of vertebrates: Homo sapiens (83.3%),Rattus rattus (8.3%), Canis familiaris, and Gallus gallus (both 4.2%).Females of Ev. lenti (1), Lu. longipalpis (9), and Ny.whitmani (1) had fed exclusively on H. sapiens, while the single female of Sc. sordellii had fed on R. rattus.Nyssomyia neivai females had fed on H. sapiens (9) and G. gallus, whereas Ev. cortelezzii females had fed on H. sapiens, C. familiaris, and R. rattus (Table 3).No instances of mixed blood meal (two or more blood sources simultaneously detected in the same sand fly) was observed in the engorged females.The cytb sequences were deposited in the GenBank database under accession numbers PP430092-PP430114.

Discussion
The findings of this study shed light on the sand fly fauna of the municipality of Iguatama, Minas Gerais (MG), Brazil, including Leishmania infection rates and blood meal sources.The predominant sand fly species identified were Lu.longipalpis, Ny. neivai, and Ny.whitmani, which together constituted the majority (88%) of the collected specimens.These species have been previously implicated as important vectors in the transmission of VL and CL.The detection of Leishmania DNA in a considerable proportion of sand fly specimens, with Le. infantum being the predominant species identified, highlights the potential role of these sand fly species in the transmission cycle of leishmaniasis.Furthermore, the identification of diverse blood meal sources suggests the opportunistic feeding behavior of the sampled sand flies.These results provide important insights into the ecology and potential transmission dynamics of leishmaniasis in the study area.
The sand fly fauna in the municipality of Iguatama was found to consist of 12 species of seven genera.Although the species accumulation curve reached its peak after the third collection, the non-linear logarithmic regression analysis indicated that the sampling effort may not have been sufficient to capture all sand fly species in the study area.This result was further  supported by the Chao1 Index, which estimated the presence of 13 species (range 11-15 species).However, the highest species richness was observed in January and May 2019, with ten and eight species collected, respectively.Overall, despite the significant presence of Lu. longipalpis, the Shannon Index indicated a high level of diversity (H = 1.27), while the Pielou Evenness Index (J = 0.51) suggested reduced evenness due to the dominance of certain species.The presence of Lu. longipalpis in the urban area of Iguatama, as well as in other cities in the Midwest Region of MG [25][26][27], highlights its role in the transmission of Le. infantum in this endemic region.The year-round presence of Ny. neivai and Ny.whitmani, important vectors of Le. braziliensis in Southeast Brazil [28], is also of epidemiological significance, considering the reported cases of human CL from 2007 to 2021 [29].Evandromyia lenti and Ev.cortelezzii were consistently collected throughout all sampling periods.These species exhibit similar behavior and adaptability to urban areas of MG [25,27,30].The detection of Leishmania DNA in Lu. longipalpis (14.1%) reinforces its significance in the parasite life cycle, as it has been shown to support late-stage infections of various Leishmania species both experimentally [31,32] and naturally [33][34][35].Natural infections and molecular detections of Leishmania, primarily Le. infantum, in Lu. longipalpis are commonly reported in Brazilian areas endemic for VL, highlighting the significance of this species in the parasite life cycle [5,25,27,35].While Lu. longipalpis is considered a permissive species, it has yet to be confirmed as a proven vector of Le. (Viannia) parasites.However, the molecular findings of parasites belonging to this subgenus draw attention to the possibility of its involvement in the transmission cycle of CL in Iguatama.
Nyssomyia neivai is a confirmed vector of Le. braziliensis, primarily in South Brazil.This sand fly has been reported carrying Le. braziliensis DNA in the states of Parana ´ [36] and Rio Grande do Sul [37].Moreover, its role in transmitting Le. infantum is also suspected, as molecular detections have identified this parasite in the states of Parana ´ [38], Santa Catarina [39], and MG in the Southeast Region of the country [40].
Several species of the genus Evandromyia, particularly Ev. lenti and Ev.cortelezzii, have been suggested as potential vectors of Leishmania in Brazil.Evandromyia lenti appears to be widely distributed, at least in the Southeast Region of the country and shares similar ecological preferences with Lu. longipalpis [41], being predominantly found in peridomestic areas [42] and domestic animal shelters in rural areas [43], as observed in Iguatama where this species was collected throughout all study periods.Similarly, Ev. cortelezzii seems to be adapting to urban and peri-urban areas in the state of MG, such as in the state capital Belo Horizonte [30].Molecular detections of Le. infantum in Ev. lenti [22,44,45] and Ev.cortelezzii [27,44,[46][47][48] raise concerns about their potential role in transmitting this parasite, although further studies on vector capacity are needed.The primary blood source for female sand flies in the municipality of Iguatama was humans, which raises epidemiological concerns, particularly with regards to the vectors Lu. longipalpis and Ny.whitmani, which exclusively fed on humans.Evandromyia cortelezzii exhibited a more eclectic feeding habit, having fed on dogs, rats, and humans, as previously reported [49].This feeding versatility, coupled with the presence of Le. infantum, reinforces the potential role of sand flies in transmitting this parasite to multiple hosts.Nyssomyia neivai was the only species that fed on chickens.Although chickens are refractory to Leishmania infection, their presence in peridomestic sites provides a valuable blood source for maintaining the sand fly population [50].Moreover, high molecular prevalence of Leishmania has been reported in sand flies that fed on chicken blood, indicating that the quality of chicken blood supports the development of Leishmania in sequential blood meals [51,52].A single engorged female of Sc. sordellii, a species known to feed on cold-blooded animals such as frogs [53], was found, and DNA sequencing revealed rats as the blood source.This finding warrants further investigation, as it represents the first report of this sand fly feeding on potential vertebrate hosts of Leishmania.Interestingly, Sc. sordellii has been found carrying Leishmania DNA in several regions of Brazil [54][55][56][57].
In conclusion, this study provides valuable insights into the sand fly fauna of the municipality of Iguatama, Minas Gerais, Brazil, including Leishmania detection rates and blood meal sources.The presence of diverse sand fly species, including the predominant vectors, Lu. longipalpis, Ny. neivai, and Ny.whitmani, highlights the potential for VL and CL occurrence in this region.The detection of Leishmania DNA, primarily Le. infantum, in sand fly specimens emphasizes their potential role in the parasite's life cycle and raises concerns about their involvement in disease transmission.Additionally, the identification of humans as the predominant blood source for sand flies, along with the feeding habits observed in other species, underscores the risk of human exposure to Leishmania parasites.These findings contribute to understanding the epidemiology of leishmaniasis in the municipality of Iguatama, underscoring the importance of vector surveillance, especially in areas where sand flies positive for Leishmania were found and where dogs seropositive for VL were previously detected [13].Geospatial analysis of human and canine cases of leishmaniasis can help identify critical points where the disease occurs.Altogether, these measures, associated with health education, should assist local authorities in mitigating disease transmission.

Fig 1 .
Fig 1. Location of the study area.The municipality of Iguatama in the Midwest Region of Minas Gerais State, Brazil, and the collection sites throughout the municipality.https://doi.org/10.1371/journal.pone.0302567.g001

Fig 2 .
Fig 2. Species accumulation curve for the sand fly fauna of the municipality of Iguatama, Minas Gerais, Brazil.The numbers 1 to 4 on the x-axis refer to collections performed from August 2018 to September 2019, while numbers 5 and 6 refer to extrapolated hypothetical collections to produce the trend curve by non-linear logarithmic regression analysis.The y-axis represents species richness.The black line indicates the number of collected species while the dotted line indicates the number of species estimated by regression analysis.https://doi.org/10.1371/journal.pone.0302567.g002